Non-small cell lung cancers (NSCLC) are aggressive tumors that are a major cause of morbidity and mortality in the United States. In the past decade, the introduction of systemic therapies such as receptor tyrosine kinase targeted inhibitors and immune checkpoint inhibitors have significantly improved patient outcomes. However, therapeutic resistance to these therapies frequently develops and is characterized by parallel signaling through other co-expressed receptor glycoproteins. We have therefore investigated the feasibility of disrupting asparagine (N) linked glycosylation, a co- and post-translational protein modification, as a strategy to block both primary and bypass glycoprotein survival signaling in NSCLC. Although this pathway is target rich and involves at least 34 gene products, pharmacologic inhibitors that regulate this process have not previously been available. We have identified a first in class small molecule inhibitor of the oligosaccharyltransferase (OST), the multi-subunit enzymatic complex that transfers glycans to elongating proteins in the endoplasmic reticulum. This inhibitor reduces OST fidelity by targeting the catalytic subunit and results in a site-specific and partial inhibition of glycosylation. In NSCLC with activation of RTK dependent oncogenic signaling, OST inhibition blocks tumor cell proliferation and couples favorably with TKIs to enhance apoptosis and xenograft tumor growth. We now propose to characterize and advance novel small molecule inhibitors of the OST with respect to catalytic subunit inhibition and effects on EGFR driven NSCLC. Because glycoprotein bypass signaling is also operative in NSCLC subtypes with FGFR1 amplification or KRAS mutation, we will define the signaling mechanisms and determine the sensitivity of these tumors to OST inhibition. This project will also investigate the effects of OST inhibition on NSCLC radiosensitivity and identify how glycoprotein dependent cellular programs mediate intrinsic and immune mediated therapeutic resistance. Together this work will advance OST inhibition as a novel treatment strategy for NSCLC with the potential of delivering an OST inhibitor that can be translated to the clinic.